Chlamydia trachomatis is the cause of trachoma, and following repeated infections the most damaging sequela is blindness. Over 15 serovariants of C. trachomatis have been described, and serovar-specific antigens are associated with protective immunity while broadly reacting antigens are associated with immunopathology. The major outer membrane protein (MOMP) of chlamydiae contains the serovar-specific antigen, however, this is an antigenically complex molecule that also contains subspecies- and species-specific antigens. Immunity to C. trachomatis eye infections is poorly understood, although animal models and human vaccine trials have demonstrated antibody mediated immune protection. The broad, long-term objectives of this proposal are 1) the evaluation of the human humoral response of trachoma tear samples to sequence-specific chlamydial epitopes and correlation of this data to clinical status, and 2) the development of a new amino acid sequence-defined immunoassay suitable for rapid seroepidemiological evaluations and assessments of immune status. These objectives will be of value for developing a basic understanding of immune mechanisms elicited during natural infection, and for developing and evaluating strategies for immune prophylactic intervention and for monitoring epidemiological intervention approaches. The specific aims are: 1) Map the antigenic determinants for the 60K outer membrane protein using recombinant DNA expression in E. coli and peptide synthesis. Expression proteins and synthetic peptides will be identified and characterized using immune human and rabbit sera. 2) Evaluation of strain diversity for the MOMP gene of serovar-specific isolates obtained in trachoma endemic areas. This is accomplished using primer extension sequencing of mRNA isolated from infected tissue cultures. 3) Definition of the molecular basis of antigenic relatedness among serovariants. The DNA sequences of each of the 15 prototype serovars for the portion of the gene which encodes the serovar-specific epitope will determined and compared. Antisera will be obtained in rabbits immunized with synthetic peptides representing these epitopes, and the specificities of the antibody reactivities will define the molecular complexities of these relationships. 4) Develop an epidemiological immunoassay using serovar-specific DNA and amino acid sequence information to clone and express sequence-defined reagents for each of the 15 serovariants. These reagents will be constructed from synthetic oligonucleotides and expressed as fusion products of glutathione transferase. One-step affinity purification of these soluble fusion proteins will provide unlimited quantities of standardized material. 5) Determination of the human humoral response to antigenic domains and sequence-specific epitopes for the MOMP and 60K proteins. Tears obtained from trachoma studies will be evaluated for immunoglobulin classes and isotypes, and their quantity, avidity, and epitopic specificities will be evaluated.